Method for the treatment of thrombocytopenia and pharmaceutical compositions useful therefor

ABSTRACT

The present invention relates generally to a method for, and pharmaceutical compositions useful in, the treatment of thrombocytopenia in a mammal by the administration of an effective amount of leukaemia inhibitory factor (LIF) and/or its derivatives and optionally in combination with one or more other cytokines.

The present invention relates generally to a method for, andpharmaceutical compositions useful in, the treatment of thrombocytopeniain a mammal by the administration of an effective amount of leukaemiainhibitory factor (LIF) and/or its derivatives and optionally incombination with one or more other cytokines.

Leukaemia inhibitory factor (LIF) was purified (1, 2) and cloned (3) onthe basis of its capacity to induce differentiation in and suppressclonogenecity of the M1 mouse myeloid leukaemic cell line (seeInternational Patent Application No. PCT/AU88/00093). LIF has comparableeffects on human HL60 and U937 cells, particularly when acting incollaboration with colony stimulating factors (4). In conventionalsemisolid cultures, LIF has no colony stimulating activity for normalmurine haemopoietic cells (5) although it stimulates the proliferationof the continuous haemopoietic cell line DA1.1a (6) and erythroid celllines from myc-transformed mouse fetal liver cells.

Receptors for LIF are present on monocytemacrophages (7) and somenon-haemopoietic cells including osteoblasts, placental and liver cells(8). LIF has been shown to possess a remarkable variety of actions: itreleases calcium from bone tissue (9), is the factor preventingspontaneous differentiation in normal embryonic stem cells (10, 11), isa molecule stimulating DA1.1a (6) cell proliferation, stimulates livercells to produce acute phase proteins (12, 13), and is a lipoproteinlipase inhibitor (14).

In initial studies, the consequences of high LIF levels were determinedin mice engrafted with FDC-P1 cells producing LIF (International PatentApplication No. PCT/U90/00092; 15, 16). Such mice developed a fatalsyndrome of body weight loss, osteosclerosis with compensatory splenicand liver extramedullary haemopoiesis, a neutrophil leukocytosis,pancreatitis, calcification in skeletal muscle, heart and liver, livernecrosis and fibrosis, thymus atrophy, adrenal cortex changes andfailure of spermatogenesis and corpora lutea formation.

The engrafted model is potentially complex due to the presence of theengrafted FDC-P1 cells. The present invention arose from experimentsattempting to overcome this complexity by injecting purified recombinantmurine LIF into mice to determine what changes could be induced byinjected LIF. Changes inter alia in blood components, marrow, spleen andperitoneal cell components, megakaryocyte and progenitor cell componentsin the marrow and spleen were analysed and it was surprisinglydiscovered that LIF caused the enhancement, stimulation and/or increasein the level of formation of megakaryocytes and/or their progenitorcells and led to an increase in platelets. The present invention,therefore, will be beneficial in the treatment of thrombocytopeniaoccurring in some acute infections, anaphylactic shock, certainhaemorrhagic diseases, anaemias, as a result of chemo- or radiotherapy,platelet- function deficient disease, chronic hepatic disorders andrenal disorders.

Accordingly, one aspect of the present invention relates to a method fortreating thrombocytopenia in a mammal which method comprisesadministering to said mammal an effective amount of leukaemia inhibitoryfactor (LIF) for a time and under conditions sufficient to enhance,stimulate and/or increase the level of formation of megakaryocytesand/or their progenitor cells and/or increase the level of platelets.

In another embodiment, LIF is administered simultaneously orsequentially with one or more other cytokines.

Another aspect of the present invention is directed to a pharmaceuticalcomposition for treating thrombocytopenia in a mammal said compositioncomprising LIF in combination with one or more other cytokines and oneor more pharmaceutically acceptable carriers and/or diluents.

Yet another aspect of the present invention relates to the use of LIFalone or in combination with one or more other cytokines and/or theirderivatives for the manufacture of a medicament for treatingthrombocytopenia by enhancing, stimulating and/or increasing the levelof formation of megakaryocytes and/or its progenitors and/or plateletsin a mammal.

In a preferred embodiment, the mammal is a human or a livestock animaland human, murine and/or a livestock animal LIF is used. Furthermore,preferred other cytokines include interleukin 3 (IL-3), thrombopoietinand/or interleukin 6(IL-6). The most preferred other cytokine is IL-3.In any event, the preferred other cytokine is of human, murine and/orlivestock animal origin.

The present invention is described in terms of the effect of LIF in miceand monkeys. This is done with the understanding, however, that thepresent invention extends to the effect of LIF in all mammals and inparticular humans and livestock animals. Accordingly, by referenceherein to the effect of LIF in mice or monkeys is meant to be applicableto the effect of LIF in mammals and in particular humans and livestockanimals.

In one embodiment, human, murine or livestock animal LIF is usedalthough the present invention extends to any mammalian LIF having thedesired activity herein described.

The term "thrombocytopenia" is used herein to denote conditions in amammal affecting levels of megakaryocytes and/or their progenitorsand/or platelets. Accordingly, the treatment of thrombocytopenia istaken to be enhancing, stimulating and/or increasing the level offormation of megakaryocytes and/or their progenitor cells and/orincreasing the level of platelets in a mammal by the administration ofan effective amount of LIF for a time and under conditions sufficient toeffect an increase in the number of megakaryocytes and/or theirprogenitors and/or platelets. Thrombocytopenia may occur following adisease condition or result from trauma or therapy and the presentinvention is not limited to any one or more causes of thrombocytopenia.Typically, thrombocytopenia will occur in some acute infectionsanaphylactic shock, certain haemorrhagic diseases, anaemias, as a resultof chemo- or radiotherapy, platelet-function deficient disease, chronichepatic disorders and renal disorders.

The present invention also extends to preventative therapy whereby LIFand optionally one or more cytokinnes are administered to prevent orreduce the likelihood of thrombocytopenia developing.

The very short serum half-life of intravenously-injected LIF indicatedthat the intraperitoneal route was more practicable in mice for ensuringsustained periods of elevated serum LIF levels. However, other routes ofadministration may be possible without departing from the scope of thepresent invention (eg. intravenous, intramuscular and subcutaneous) andall such routes are encompassed herein. Irradiated mice engrafted withLIF-producing cells developed serum levels of up to 10³ units/ml andsome developed organ changes within 14 days (15, 16). The initialschedule chosen to attempt to document in vivo effects of injected LIFwas 2 μg three times daily for 14 days which would achieve LIF levelsabove 10³ units/ml for several hours after each injection. The extremeside effects encountered suggest that these were toxic. The unexpectedand unanticipated changes observed in the work leading to the presentinvention such as those in platelet levels were not recognised initiallyand not all mice were analysed for these changes.

In accordance with the present invention, it has been discovered that aclear elevation of progenitor cells is induced in the spleen by theinjection of LIF, alone or in combination with one or more othercytokines, and this includes rises in megakaryocyte progenitors. Whilethe progenitor cell changes do not result in observable increases inmature neutrophils, monocytes or eosinophils, they are associated with arise in megakaryocyte numbers, followed by a rise in blood plateletlevels. The magnitude of the megakaryocyte and platelet rises induced byLIF is alone or in combination with one or more other cytokines equal toor greater than those induced by IL-3 (18), thrombopoietin (19) or IL-6alone (20, 21) indicating the potential clinical use of LIF to treatthrombocytopenia by increasing the level of megakaryocytes and/or theirprogenitor cells and/or platelets. In this context, it is of interestthat reduced doses of LIF are still able to induce changes inmegakaryocyte and platelet levels without toxic effects as assessed bybehavioural changes or body or thymus weight loss. A particularlyeffective combination is LIF and IL-3.

While LIF appears to have no colony stimulating activity in conventionalsemisolid cultures of unfractionated mouse marrow cells or purifiedprogenitor cells (Hilton DJ, Nicola NA and Metcalf D, unpublished data),the present invention demonstrates that megakaryocytes do express LIFreceptors. Although it is not the intention to limit the presentinvention to any one theory behind the mode of action, the stimulatingeffects of LIF on megakaryocyte and platelet formation could representdirect effects possibly in association with some other factors. LIFinduces rises in megakaryocyte progenitor cells and megakaryocytesbefore rises in platelet levels, suggesting that the observed rises inplatelets are based on the increased formation of megakaryocytes and notmerely the induced release of platelets from existing megakaryocytes.Furthermore, the effects of injected LIF on progenitor cell levels,megakaryocyte formation and platelet levels described herein are incontrast to the apparent inactivity of LIF on normal haemopoietic cellsin vivo. Another possibility may be that LIF may interact with or elicitproduction of some other megakaryocyte stimulatory factors.

Accordingly, the present invention contemplates a method of treatingthrombocytopenia in a mammal which method comprises administering tosaid mammal an effective amount of LIF alone or in combination with oneor more other cytokines for a time and under conditions sufficient toincrease the numbers of megakaryocytes and/or their progenitors and/orplatelets.

Preferably the mammal is a human or a livestock animal although thepresent invention is not so limited. Furthermore, the route ofadministration is preferably by interperitoneal, intravenous,intramuscular or subcutaneous administration (eg injection) but otherroutes may be equally applicable with only minor modification to themethod as contemplated herein. The effective amount of LIF will dependon the mammal and the condition to be treated. For example, in mice, thefrequency of megakaryocytes was increased in the spleen 2-5 fold afteri.p. injection one to three times daily with 2 μg LIF for 3-14 days.However, the amount required to be administered to the mammal will needto be non-toxic. Hence, in mice, for example, a dose of 200 ng or lowergiven one to three times daily for 14 days while causing a slightlysmaller increase in megakaryocytes and platelets compared to the neartoxic dosages was never-the-less effective and, importantly, non-toxic.In general, the effective amount of LIF and, where used, the cytokinewill be 0.01 to 10,000 μg/kg and preferably, 1 to 1000 μg/kg bodyweight.

Use herein of the term "livestock animal" is intended to include suchanimals as sheep, pigs, goats, horses, donkeys and cows and furtherextends to cats and dogs.

The method of the present invention further contemplates thesimultaneous or sequential administration of LIF with one or more othercytokines. Such cytokines include, but are not limited to, IL-3thrombopoietin and/or IL-6. In a most preferred embodiment, LIF is givenwith IL-3. Such a mode of administration encompasses the administrationof a single composition comprising both LIF and the cytokine(simultaneous administration) or the administration of two separatecompositions, one containing LIF and the other containing one or moreother cytokines (sequential administration). The present inventionextends to the use of more than one cytokine in separate compositions orin a single composition. Furthermore, the present invention contemplatesthe use of LIF and the cytokine in any order. In another embodiment onecytokine (including LIF) may be given by direct injection while theother cytokine is administered by, for example, a drip. In sequentialadministration the present invention is not limited to any time periodbetween the administration of the two compositions. Preferably, however,the time difference would be less than 72 hours.

In all of the above cases, the present invention also extends to the useof derivatives, homologues and/or analogues of LIF and the othercytokines. By "derivative" and "analogue" are meant recombinant,chemical or other synthetic forms of LIF or other cytokine and/or anyalterations such as addition, substitution and/or deletion to the aminoacid sequence component of the molecule or to the carbohydrate or otherassociated molecule moiety (if present) of LIF or other cytokine,provided the derivative possesses megakaryocyte, megakaryocyteprogenitor and/or platelet stimulating activity. Preferably, the LIF isof human, murine or livestock origin but the present invention is notnecessarily limited thereto. Use herein, therefore, of the terms "LIF"and "cytokine" is intended to encompass any one or more of theirderivatives, homologues or analogues including naturally occurring(natural) or recombinant or synthetic forms.

In accordance with the present invention, the LIF and one or more othercytokines (eg IL-3) may be from the same or different mammalian species.

The present invention further extends to pharmaceutical compositionscomprising LIF in combination with one or more other cytokines and/ortheir derivatives and one or more pharmaceutically acceptable carriersand/or diluents. Such pharmaceutical compositions will be useful inenhancing, stimulating and/or increasing the level of formation ofmegakaryocytes and/or its progenitor cells and/or platelet cells.

The method and pharmaceutical compositions described in accordance withthe present invention will be useful in the treatment inter alia ofthrombocytopenia which occurs in some acute infections, anaphylacticshock, certain haemorrhagic diseases, leukaemia anaemias, as a result ofchemo- or radiotherapy, platelet-function deficient disease, chronichepatic disorders and renal disorders. Furthermore, the presentinvention extends to the use of LIF and/or its derivatives alone or incombination with one or more other cytokines and/or their derivativesfor the manufacture of a medicament for enhancing, stimulating and/orincreasing the level of formation of megakaryocytes and/or itsprogenitors and/or platelets in a mammal, and in particular humans andlivestock animals, such as in the treatment of thrombocytopenia.

The presence of LIF after administration by i.p. injection had otheraffects as outlined below.

The haemopoietic changes observed in LIF-injected mice (see Example 2)had a pattern which indicated that LIF has either direct or indirectactions affecting certain haemopoietic populations. Injections of 2 μgLIF in mice, for example, failed to reproduce the characteristicneutrophil leukocytosis seen in the engrafted model (16) but didreproduce other changes seen in such mice including reduced marrowcellularity with selective loss of marrow lymphocytes, depression ofspleen lymphocyte populations with increased splenic erythropoiesis andmarked thymus atrophy due to loss of cortical lymphocytes.

Furthermore, early effects of high doses of LIF were a hyperactive stateand body weight loss, the latter based on a reduction in subcutaneousand abdominal fatty tissue. The hyperactive state may be related to theability of LIF to switch autonomic nerve signalling from adrenergic tocholinergic mode or be related to the hypercalcemia (22). The selectiveloss of body fat may be based on the lipoprotein lipase inhibitoryactivity of LIF (14) which may prevent the transport of lipid into fatcells. The increased erythrocyte sedimentation rate noted inLIF-injected mice was apparent within 6 hours of a single injection andmay be due to the ability of LIF to induce the production by liver cellsof acute phase proteins (12, 13) likely to influence erythrocytesedimentation.

No abnormalities were noted in other organs of LIF-injected mice otherthan minimal calcification in the myocardium and this was seen only inmice injected with the highest doses of LIF.

One of the most striking changes in LIF-engrafted mice was the excessosteoblast activity and new bone formation occurring particularly in thesternum and end of the long bones (15, 16). Marked changes of thisnature were not observed in LIF-injected mice but LIF injectionspossibly induced some thickening of the cortex of sternal bone segments.After the intravenous injection of ¹²⁵ I-labelled LIF, labelling ofmarrow osteoblasts was observed so injected LIF has access to thesecells and LIF may therefore have direct actions on osteoblasts and newbone formation. In this context, initial experiments injecting LIF for 3days to preirradiated, but not normal mice, has produced a definiteincrease in reticulum in the bone marrow, a change seen in dramatic formin the marrow of mice engrafted with LIF-producing cells. The ability ofeven low doses of LIF to elevate serum calcium levels may be ofsignificance for an action of LIF in promoting new bone formation.

The present invention is further described by the following non-limitingFigures and Examples.

In the Figures:

FIG. 1 is a graphical representation showing serum LIF levels in DBA/2mice following the intraperitoneal injection of 2 μg LIF. Each pointrepresents serum levels from a different mouse.

FIG. 2 is a graphical representation showing the loss of body weight inDBA/2 and C3H/HeJ mice injected three times daily with 2 μg LIF. Notethat body weight loss is restricted to the first week.

FIG. 3 is a graphical representation showing the elevation of serumcalcium/albumin ratios in C3H/HeJ mice injected three times daily for 14days with 2 μg LIF.

FIG. 4 is a pictorial representation showing the increase inmegakaryocytes in the spleen of a DBA/2 mouse injected with 2 μg LIF 3times daily for 14 days (A) versus spleen from a control mouse injectedwith FCS/saline (B). Haematoxylin and eosin (×250).

FIG. 5 is a graphical representation showing the increase in thefrequency of progenitor cells in the marrow and spleen of mice injectedwith 2 μg LIF 3 times daily for 14 days (o) versus frequency in controlmice injected with FCS/saline (o). Each point represents data from anindividual animal.

FIG. 6 is a graphical representation showing enhancement ofmegakaryocyte colony formation in IL-3 stimulated cultures of 50,000 ofC3H/HeJ marrow cells by inclusion of 1000 units per ml of LIF. Eachpoint represents mean values from duplicate cultures.

FIG. 7 is a graphical representation showing pooled data from 18separate experiments on megakaryocyte colony formation in cultures of50,000 C3H/HeJ marrow cells stimulated either by 500 units per ml IL-3plus 0.1 ml saline or 500 units per ml IL-3 plus 1000 units per ml LIF.Each point represents the number of colonies in a single culture dish.

FIG. 8 is a graphical representation showing the distribution analysisof the absolute number of megakaryocytes in colonies wholly or partlycomposed of megakaryocytes, sequential analysis of 444 coloniesstimulated by 500 units per ml IL-3 plus 0.1 ml saline and 565 coloniesstimulated by 500 units per ml IL-3 plus 1000 units per ml LIF.

FIG. 9 is a graphical representation showing the frequency distributionof grain counts in autoradiographs of marrow megakaryocytes binding ¹²⁵I-LIF with or without an excess of unlabelled LIF.

FIG. 10 is a graphical representation showing the the platelet responseto rhLIF in Rhesus monkeys.

FIG. 11 is a graphical representation showing the platelet response torhLIF in Rhesus monkeys.

EXAMPLE 1 Materials and Methods

Mice

Mice used were specific pathogen-free 2 to 3 month old females of thestrain DBA/2 (the strain previously used as recipients of LIF-producingFDC-P1 cells as described in International Patent Application No.PCT/AU90/00092) and the endotoxin hyporesponsive strain, C3H/HeJ, thelatter to minimise the possibility that any observed changes might beascribable to endotoxin.

Recombinant LIF

Recombinant murine LIF was produced using the GEX bacterial expressionsystem and purified to homogeneity as previously described(PCT/AU88/00093). The specific activity of the LIF was approximately 10⁸units/mg protein as assayed on M1 leukaemic cells (50 units/ml is theconcentration of LIF inducing the differentiation of 50% of M1 coloniesin agar cultures of 300 M1 cells--see PCT/AU88/00093).

The recombinant LIF was dissolved in 5% (v/v) fetal calf serum(FCS/saline) so that each dose injected was in a volume of 0.2 ml.Control mice were injected with 0.2 ml of the same 5%(v/v) FCS/salinediluent batch. Two separate preparations of LIF were used and twodifferent batches of FCS/saline used as diluent. All preparations wereassayed by the Limulus amoebocyte lysate assay and the injection volumesof 0.2 ml of LIF in FCS/saline or 0.2 ml FCS/saline were found tocontain 0.1-0.2 ng endotoxin, indicating that the endotoxin content ofthe material probably originated from the FCS used.

Injections

Mice were injected one to three times daily for up to 14 days with 0.2ml of LIF or FCS/saline, weighed at intervals then analysed in detail onthe morning after completion of the last day of injections.

Cultures

All cultures were performed using 35 mm Petri dishes containing 50,000bone marrow cells from 2 months old C3H/HeJ mice in 1 ml of agar-mediumwith a final concentration of 20% (v/v) fetal calf serum in 0.3% (w/v)agar.

Cultures were incubated at 37° C. in a fully humidified atmosphere of10% (v/v) CO₂ in air. After 1 week of incubation, colony counting wasperformed at ×35 magnifications then all cultures fixed using 1 ml of2.5% (w/v) glutaraldehyde in 0.9% (w/v) saline. After floating theintact cultures onto glass slides, the cultures were air-dried andstained for acetylcholinesterase, then with Luxol-Fast-Blue andhaematoxylin. Using coded slides, megakaryocyte colonies (defined asclones containing two or more acetylcholinesterase-positive cells) wereenumerated and total cell counts performed ofacetylcholinsterase-positive cells in each colony.

All stimuli tested were purified murine recombinant factors produced inthis laboratory using bacterial or yeast expression systems: specificactivities for LIF, IL-3, GM-CSF, G-CSF and M-CSF were all 10⁸ Units/mg.

Autoradiography

Purified recombinant murine LIF was labelled with ¹²⁵ I using methodsdescribed previously(3). Adult mice were injected intravenously with 10⁸counts/min ¹²⁵ I-LIF and killed 1 hour later. Tissues were fixed in 10formal saline, 5μ sections prepared and dipped in Kodak N2 emulsion.After exposure for 3 months, the slides were developed and stained withhaematoxylin and eosin. For in vitro studies, spleen and marrowsuspensions were incubated for 1 hour at 37° C. with ¹²⁵ -LIF (100,000counts/min) with or without a 20-fold excess of unlabelled LIF. Thecells were washed, cytocentrifuge preparations fixed using 2.5% (w/v)glutaraldehyde. After dipping and exposure, the preparations werestained with May-Grunwald Giemsa.

Observations

Mice were anaesthetised and orbital plexus blood used for white cell,haematocrit and platelet estimations. Mice were exsanguinated from theaxillary vessels and the serum diluted 1:4 for further analysis.Peritoneal cavity cells were collected using 2 ml of 5% (v/v)FCS/saline, organs were weighed and total femur cell counts performed.Cytocentrifuge preparations were made from peritoneal, spleen and marrowcells and stained with May-Grunwald/Giemsa. All preparations were scoredusing coded slides. Organs were fixed in 10% (v/v) formal saline,sectioned then stained with haematoxylin and eosin for reticulum. Spleenand marrow cell suspensions were cultured to determine the frequency ofprogenitor cells using 1 ml agar-medium cultures of 25,000 cellsstimulated by a mixture of 400 units GM-CSF and 400 units IL-3 (17). Atday 7, colony counts were performed and the cultures mixed with 1 ml of2.5% (w/v) glutaraldehyde then stained sequentially foracetylcholinesterase then Luxol Fast blue and haematoxylin, differentialcolony counts were performed at ×200 magnification on codedpreparations.

Erythrocyte sedimentation was measured in heparinized capillaryhaematocrit tubes using a 50 mm column of blood. Acceleratedsedimentation of red cells was noted within 10 min with blood fromLIF-injected mice but measurements were made routinely at 2 hours. Forsimplicity, the figures were transformed arithmetically to mmsedimentation per 100 mm column.

Serum calcium and albumin estimations were performed using 1-4 dilutedserum.

Megakaryocyte cell counts were performed at ×400 magnifications fromsections of spleen and sternal marrow segments. The area surveyed wasdetermined from camera lucida drawings and the figures expressed asmegakaryocytes X area⁻¹ X organ weight for spleen or megakaryocytes Xarea⁻¹ X 100 for individual sternal marrow segments.

Statistical Analysis

All data were analysed using the student T test to establish thestatistical significance of observed differences.

EXAMPLE 2 Effect of LIF

The mean serum LIF concentrations in mice engrafted with LIF-producingFDC-P1 cells were 1000 units/ml (15, 16). To determine if comparableconcentrations could be achieved by the injection of LIF, studies wereperformed on the serum half-life of LIF injected intravenously orintraperitoneally. Intravenous injection of 2 μg of LIF resulted in avery short serum half-life with a second phase of 8-9 minutes. Howeverthe intraperitoneal injection of 2 μg LIF resulted in a more sustainedelevation of serum LIF levels which exceeded 1000 units/ml forapproximately 3 hours (FIG. 1). On this basis, initial injections wereperformed three times daily at 8.00 am, 2.00 pm and 5.00 pm using 2 μgLIF injected intraperitoneally. In subsequent experiments lower doses ofLIF were used and the number of injections per day varied from 1-3.

General Observations

The dose of 2 μg LIF three times daily appeared to be close to the toxiclimit since deaths occurred at day 3 of the smallest of 16 DBA/2 mice,of 4 of 8 possibly abnormal C3H/HeJ mice between days 5 and 9, and ofthe smallest of another group of 8 C3H/HeJ mice on day 5.

At this dose level, a uniform effect of injected LIF in mice of bothstrains in five separate experiments was an initial weight loss evidentby day 2 and progressive during the first week of injections but with nofurther weight loss during the second week (FIG. 2). Accompanying theweight loss was a curious state of hypermobility and irritability of themice with the hair on the back and particularly on the back of the neckbeing erect. No fighting was exhibited. The LIF-injected mice alsoexhibited difficulty in recovering from the anaestheticused--methoxyfluorane.

No deaths or weight loss occurred in mice injected with 200 ng or lowerdoses of LIF, given 1-3 times daily for 14 days and no irritability ofbehaviour was observed at these dose levels.

Blood Changes

The changes observed at day 15 in mice injected with 2 μg LIF threetimes daily are summarised in Table 1. No significant changes occurredin total white cells but a small fall in haematocrit was observed inLIF-injected mice. A notable finding was an increase in platelet levelsof most 100% in LIF-injected mice. Platelet levels were not elevated at6 and 24 hours after a single injection of 2 μg LIF or after 3 days ofinjections of 2 μg LIF given three times daily (Tables 1,2).

Another feature of blood samples from mice injected with 2 μg LIF was anacceleration of erythrocyte sedimentation. In other experiments,accelerated erythrocyte sedimentation was noted at 6 and 24 hoursfollowing a single injection of 2 μg LIF and at 14 days with as low adose of LIF as 200 ng given once daily (Table 2).

As shown in the typical example in FIG. 3, serum calcium levels wereelevated in mice injected with 2 μg LIF for 14 days, the rises averaging30% above values in control-injected mice. Elevated serum calcium levelswere observed with as low a dose as 20 ng given once daily for 14 days(Table 2). In mice injected with 2 μg LIF three times daily elevatedcalcium levels were not present after 6 hours but were present after 3days of injections.

Marrow, Spleen and Peritoneal Cell Changes

Table 3 summaries data from DBA/2 mice injected with 2 μg LIF threetimes daily for 14 days. For brevity, similar data from C3H/HeJ micehave not been detailed.

A uniform finding in mice of both strains was a fall of approximately40% in total marrow cell numbers with a significant fall in thepercentage of lymphoid cells and a small, but significant, rise in thepercentage of mature granulocytes.

A minor weight increase was observed in the spleen of LIF-injected miceand, as shown in Table 3, in LIF-injected mice there was a significantfall in the percentage of lymphocytes and a significant rise in thepercentage of nucleated erythroid cells and mature granulocytes.

Some variability was encountered between experiments in the totalnumbers of peritoneal cells in LIF-injected mice with no consistenttrend being obvious. Injection of FCS/saline induced the appearance ofsignificant numbers of eosinophils, presumably an immune response forthe repeated injection of foreign protein and only becoming evidentduring the second week of injections. This eosinophil response wassignificantly lower or absent in LIF-injected mice. Conversely,LIF-injected mice exhibited a significant elevation in the percent oflymphocytes in peritoneal populations.

In all of the above parameters, the changes observed in LIF-injectedC3H/HeJ mice were identical in direction and also statisticallysignificant but usually were slightly smaller in magnitude.

In DBA/2 mice injected with 200 ng LIF, 1 to 3 times daily comparablebut less marked changes in bone marrow cellularity were observed butinterestingly spleen enlargement was more evident that with higher LIFdoses (Table 2).

Megakaryocyte Changes

Because megakaryocytes were not adequately represented in cytocentrifugepreparations, counts of megakaryocyte numbers were made from sections ofthe spleen and sternal marrow segments. In both strains injected threetimes daily with 2 μg LIF for 14 days the frequency of megakaryocyteswas significantly increased in the spleen (2-5-fold) (Table 4, FIG. 4)and significant elevation so lesser magnitude were observed in thesternal marrow.

Significant elevations of spleen megakaryocyte numbers were stilldetectable (3-fold) with as little as 20 ng LIF injected once daily for14 days and megakaryocyte numbers were elevated in the spleen within 3days of injecting 2 μg LIF three times daily (Table 2).

Progenitor Cell Changes in Marrow and Spleen

The frequency of progenitor cells (exclusive of megakaryocyteprogenitors) was significantly higher in the bone marrow of DBA/2 andC3H/HeJ mice injected with 2 μg LIF three times daily for 14 days thanin the marrow of control-injected mice (FIG. 5). However, when thesevalues were corrected for the fall in total marrow cellularity, totalprogenitor cell numbers were essentially unaltered by LIF injections.

In contrast, a marked rise in the frequency of progenitor cells wasobserved in the spleen of both strains injected with LIF. Since theoverall size of the spleen was slightly increased, this indicates a risein the absolute numbers of progenitor cells. Differential colony countsrevealed no differences between LIF-injected and control mice in therelatively frequencies of the various subsets of progenitor cells:granulocytic, granulocyte-macrophage, macrophage, eosinophil, erythroidand mixed-erythroid progenitors.

The frequency of megakaryocyte colonies is not shown in the data in FIG.5 and, to determine their frequency, counts were performed independentlyon acetylcholinesterase-stained cultures. The frequency of megakaryocyteprogenitors was significantly higher in the marrow of LIF-injected micethan in control mice and 10-fold higher in the spleen of LIF-injectedmice that in control mice (Table 4). When corrected for absolute cellnumbers, there was little absolute rise in megakaryocyte progenitors inthe marrow but in the spleen the rises were at least 10-fold.

Other Changes in LIF-Injected Mice

In mice injected with 2 μg LIF three times daily for 14 days,examination showed that the loss of body weight was ascribable tocomplete loss of subcutaneous and abdominal fat, a change that was alsoevident after only 3 days of injections. Liver and kidney weights wereunaltered, indicating that the weight loss was not a true cachexia.

Mice receiving these doses of LIF exhibited pronounced thymus atrophy(Table 2) due to complete loss of cortical lymphocytes. No thymus weightloss was observed in mice injected with lower doses of LIF.

The liver showed no evidence of infiltration by haemopoietic cells, noincrease in resident Kupffer cells, and no calcification. However, inmice receiving 2 μg LIF three times daily for 14 days there was acurious reduction in the number of parenchyma cell nuclei per unit area(eg in DBA/2 mice from 20±2 in control mice to 16±3 in LIF-injectedmice, 0.01 (P<0.02)) with a corresponding increase in the area ofnucleus-free cytoplasm. No pyknosis of liver cell nuclei was observed.

Small foci of calcium deposition was observed in the myocardium of 8 of11 DBA/2 mice injected with 2 μg LIF versus 4 of 12 control mice, aslight but not significant difference.

No histological abnormalities were noted in the pancreas, ovaries,adrenal cortex or skeletal muscle comparable with those present in miceengrafted with LIF-producing cells.

In view of the marked excess new bone formation in mice engrafted withLIF-producing FDC-P1 cells (15,16), an analysis was made of the femur,tibia and sternum. No obvious new bone formation was observed in thefemur or tibia as assessed by unusual trabeculae formation. However,analysis of sternal segments indicated a significant thickening of thebone cortex in LIF-injected DBA/2 mice. In mice injected with 2 μg LIF 3times daily for 14 days the area occupied by bone cortex was 30.4±4.2%of the total area of sternal segments versus 23.1±5.6% in control mice(P<0.01). However, in C3H/HeJ mice given the same LIF dosage the figureswere 23.1±5.3% versus 21.4±4.4, a not significant differences.

EXAMPLE 3 Effect of LIF and II-3

Seven-day cultures of marrow cells containing 100 Units/ml of LIF showedno megakaryocyte or other colony formation and no surviving singlemegakaryocytes. In cultures containing combinations of LIF (1000Units/ml) with 1000 Units/ml of GM-CSF, G-CSF or M-CSF, again nocolonies containing megakaryocytes were observed nor were there singlesurviving megakaryocytes.

In marrow cultures containing from 125 to 1000 Units/ml of IL-3,megakaryocyte colony formation was observed. These colonies usually wereof two types--those containing small numbers of large dispersedmegakaryocytes or large colonies containing acetylcholine-positive cellsof various sizes. Less frequently, mixed colonies were observedcontaining megakaryocytes together with cells of other lineages andtypically in these colonies the number of acetylcholinesterase-positivecells was relatively small. Inclusion of 1000 Units of LIF in suchcultures increased the number of megakaryocyte colonies developing withall concentrations of IL-3 (FIG. 6). Data on megakaryocyte colonynumbers from 18 separate experiments using 500 Units of IL-3 indicatedthat in individual cultures, the number of megakaryocyte colonies variedwidely. In the same 18 experiments, in cultures containing 500 Units ofIL-3 plus 1000 Units/ml of LIF, a significant overall increase inmegakaryocyte colony numbers was observed (FIG. 7) (t-4.43, P<0.01)despite the variation between individual cultures.

Combination of LIF with IL-3 had no influence on the number or size ofgranulocyte-macrophage colonies developing in these cultures comparedwith cultures containing IL-3 alone. To document that this enhancedcolony formation actually resulted in the production of moremegakaryocytes, total megakaryocyte numbers were determined by countingcolony megakaryocytes in the entire culture dish in fourteen experimentsusing 500 Units IL-3 alone or in combination with 1000 Units of LIF. Incounts on 50 unselected cultures of each type, the addition of LIFsignificantly increased the total number of megakaryocytes developingper culture from 183±122 to 300±185 (±SD) (t=3.79, p<0.01).

The frequency distribution of megakaryocyte numbers in individualcolonies was analysed to determine whether LIF might exert a selectiveeffect on the size of small colonies of mature cells or on the largercolonies containing megakaryocytes at varying stages of maturation. Thehistogram in FIG. 8 shows the frequency distribution of coloniescontaining small or large numbers of megakaryocytes. The addition of LIFdid not appear to result in an exclusive increase in frequency of anyparticular subset of megakaryocytecontaining colonies.

EXAMPLE 4 Receptors for LIF on Megakaryocytes

Marrow suspensions enriched for megakaryocytes were incubated in vitrowith ¹²⁵ I-labelled LIF with or without a 20-fold excess of unlabelledLIF. As shown in FIG. 9, labelling was exhibited by approximately 85% ofmegakaryocytes. In the presence of an excess of unlabelled LIF,labelling was significantly reduced by not eliminated. This suggeststhat part of the observed labelling may have been non-specific and inthis context it was evident that damaged megakaryocytes exhibitedprominent labelling that was not blocked by unlabelled LIF. It was alsoevident that mature megakaryocytes exhibited higher grain counts thanless mature cells with basophilic cytoplasm (mean grain count for maturecell=80±50 versus immature cells 11±14 grains per cell).

EXAMPLE 5 Effect of LIF on Blood Platelet Number in Monkeys

Materials and Methods

Animals

Eight adult Rhesus monkeys, Macaca mulatta of both sexes, approximately6 to 10 years old, weighing 6 to 11 kg, were individually housed.Monkeys were provided with 10 changes per hour of fresh air conditionedto 23° C.±2° C. with a relative humidity of 60% ±10. They weremaintained in a 12 hour light/dark cycle and were provided with tapwater ad libitum and commercial primate chow and fruits.

    ______________________________________                                        LIF dosages and treatment schedule                                                                  Dose of rhLIF                                           Monkey No.    Sex     (μg/kg/day 0-13)                                     ______________________________________                                        B62           male    50                                                      B10           female  50                                                      D13           male    10                                                      645           female  10                                                      C78           male    2                                                       V143          female  2                                                       C48           male    --                                                      D41           female  --                                                      C87           male    --                                                      ______________________________________                                    

Administration of rhLIF

Frozen stock solutions of rhLIF provided by Dr. Nicos Nicola, (TheWalter and Eliza Hall Institute of Medical Research, Melbourne,Australia), were divided into the daily amounts and stored at -70° C.,again. The daily amounts were thawed and diluted with 4 ml salinesupplemented with 0.5% monkey serum. Daily doses of the cytokine weredivided into two administrations and injected subcutaneously (s.c.)between 8 and 9 am and 4 and 5 pm. Samples for determining biologicalactivity were retained at the beginning and at the end of the treatmentperiod. Control monkeys received s.c. injections of non-pyrogenic salinesupplemented with 0.5% monkey serum.

Hematologic Examinations

Peripheral blood was collected for hematologic examinations inEDTA-coated tubes before treatment began, either daily or at two dayintervals during the treatment period, and three times a week during thepost-treatment period.

Parameters measured included the total count of red blood cells (RBC),white blood cells (WBC), platelets, and determination of hemoglobin andhematocrit (Sysmex 2000, TOA: Tokyo, Japan). Differential blood cellcounts were established as normal for Rhesus monkeys (23) on theexamination of 200 cells of Giemsa-stained blood smears by twoindependent observers.

Results

Elevation of Blood Platelet Counts

As shown in FIG. 10, one of the animals receiving a daily dose of 2 μgLIF/kg showed a rise in blood platelet counts at the end of the two weektreatment period. Maximum platelet counts 1.4 fold elevated above normallevels were determined around day 5 after termination of the LIFadministration. The other animal in the 2 μg dose group showed a minorrise in platelet counts. The two monkeys receiving a daily dose of 10 μgLIF/kg for two weeks responded with a maxiumum rise in platelet countsof approximately 1.5 fold above basal levels. As shown in FIG. 11,animals treated with a daily dose of 50 μg LIF/kg responded with anearlier rise in platelet counts beginning on day 2-3 after theinitiation of treatment, with maxiumum levels in the range of 2-3 foldabove normal levels at the end of the administration period.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

                                      TABLE 1                                     __________________________________________________________________________    Changes in the Peripheral Blood After the Injection of LIF                               DBA/2                    C3H/HeJ                                   Parameter  Normal (n = 4)                                                                        FCS/Sal (n = 12)                                                                        LIF (n = 11)                                                                         Normal (n = 4)                                                                         FCS/Sal (n                                                                             LIF (n                  __________________________________________________________________________                                                          = 7)                    WBCs/μL 5,760 ± 1,810                                                                      5,890 ± 230                                                                          5,040 ± 1,260                                                                     3,950 ± 1,300                                                                       3,010 ± 1,140                                                                       3,410 ± 960          Neutrophils/μL                                                                        840 ± 230                                                                          780 ± 410                                                                            1,120 ± 430                                                                       1,070 ± 710                                                                         700 ± 320                                                                           980 ± 380            Hematocrit %                                                                             46 ± 1                                                                             46 ± 2 42 ± 3                                                                            45 ± 1                                                                              43 ± 1                                                                              41 ± 3               Platelets/μL × 10.sup.6                                                         1.01 ± 0.08                                                                        1.09 ± 0.10                                                                          1.82 ± 0.13                                                                       0.80 ± 0.10                                                                         0.92 ± 0.15                                                                         1.78 ± 0.19          ESR        1.4 ± 0.3                                                                          --        --     1.0 ± 0.7                                                                           1.2 ± 0.7                                                                           4.1                     __________________________________________________________________________                                                          ± 1.0                 Mice were examined on day 15 after the injection of 2 μg LIF three         times daily for 14 days. Mean values ± SDs.                                Abbreviation: ESR, erythrocyte sedimentation rate.                       

                                      TABLE 2                                     __________________________________________________________________________    Effects of Dose and Duration of LIF Injections on Responses Observed                                                                  Ratio                                                                         Calcium/                                                                      Albumin                                      Thymus      Spleen               (mmol/L/              Mouse Dose Schedule of                                                                        Body   Weight                                                                             Spleen Mega-   Platelets    mgmL .sup.1                                                                   ×               Strain                                                                              LIF or FCS/Sal                                                                          Weight (g)                                                                           (mg) Weight (mg)                                                                          karyocytes                                                                            (×10.sup.6 /μL)                                                             ESR   10)                   __________________________________________________________________________    DBA/2 2 μg × 3 14 d                                                                  20.6 ± 0.8                                                                         10 ± 5*                                                                        107 ± 14                                                                           442 ± 134*                                                                         1.82 ± 0.13*                                                                     --     0.95 ± 0.04*            FCS/Sal × 3 14 d                                                                  20.5 ± 1.2                                                                        35 ± 2                                                                           92 ± 14                                                                          93 ± 42                                                                            1.09 ± 0.10                                                                       --    0.82 ± 0.09              200 ng × 3 14 d                                                                   21.5 ± 1.1                                                                        36 ± 6                                                                          149 ± 5*                                                                          286 ± 30*                                                                           1.39 ± 0.17*                                                                     4.2 ± 1.3                                                                        0.85 ± 0.05              FCS/Sal × 3 14 d                                                                  20.6 ± 0.7                                                                        34 ± 2                                                                           92 ± 20                                                                          61 ± 15                                                                            0.96 ± 0.08                                                                       2.7 ± 1.4                                                                        0.77 ± 0.05              200 ng × 2 14 d                                                                   21.5 ± 0.8                                                                        37 ± 5                                                                           146 ± 10*                                                                        226 ± 89*                                                                           1.20 ± 0.08*                                                                      4.0 ± 1.1*                                                                      0.85 ± 0.03              FCS/Sal × 2 14 d                                                                  20.7 ± 0.9                                                                        40 ± 4                                                                          99 ± 9                                                                            83 ± 23                                                                            0.88 ± 0.09                                                                       1.2 ± 0.3                                                                        0.79 ± 0.05              200 ng × 1 14 d                                                                   19.3 ± 1.3                                                                         51 ± 10                                                                        130 ± 24                                                                          82 ± 39                                                                             1.15 ± 0.17*                                                                     4.5 ± 1.7                                                                        1.05 ± 0.08              20 ng × 1 14 d                                                                    19.0 ± 1.0                                                                        47 ± 3                                                                          123 ± 24                                                                          108 ± 38*                                                                          0.93 ± 0.07                                                                       2.0 ± 1.6                                                                        1.02 ± 0.06              FCS/Sal × 1 14 d                                                                  19.3 ± 0.7                                                                        50 ± 6                                                                          100 ± 26                                                                          31 ± 11                                                                            0.79 ± 0.09                                                                       2.8 ± 1.2                                                                        0.91 ± 0.02              10 ng × 1 14 d                                                                    21.3 ± 0.4                                                                        33 ± 1                                                                          101 ± 12                                                                          123 ± 40                                                                           0.91 ± 0.11                                                                       1.6 ± 0.9                                                                        0.82 ± 0.07              2 ng × 1 14 d                                                                     21.6 ± 1.1                                                                         43 ± 11                                                                         97 ± 17                                                                          111 ± 44                                                                           0.98 ± 0.14                                                                       1.0 ± 0.4                                                                        0.71 ± 0.06              FCS/Sal × 1 14 d                                                                  21.8 ± 1.5                                                                        40 ± 8                                                                          103 ± 7                                                                           93 ± 14                                                                            0.87 ± 0.17                                                                       1.2 ± 0.3                                                                        0.78 ± 0.03        C3H/HeJ                                                                             2 μg × 3 14 d                                                                   16.2 ± 1.9*                                                                        9 ± 2*                                                                         93 ± 31                                                                           148 ± 107*                                                                         1.78 ± 0.19*                                                                      4.1 ± 1.0*                                                                       0.93 ± 0.13*            FCS/Sal × 3 14 d                                                                  19.7 ± 1.4                                                                        30 ± 4                                                                          101 ± 20                                                                          76 ± 48                                                                            0.92 ± 0.15                                                                       1.2 ± 0.7                                                                        0.74 ± 0.03              2 μg × 3 3 d                                                                   16.8 ± 1.6                                                                         14 ± 5*                                                                         94 ± 31                                                                          131 ± 72                                                                           0.82 ± 0.05                                                                        5.7 ± 2.4*                                                                      0.98 ± 0.09              FCS/Sal × 3 3 d                                                                   18.4 ± 0.3                                                                        30 ± 6                                                                           94 ± 18                                                                          49 ± 22                                                                            0.87 ± 0.11                                                                       1.9 ± 0.9                                                                        0.84 ± 0.02              2 μg × 1 6 h                                                                   --     --   --     --      0.95 ± 0.07                                                                       1.8 ± 0.6                                                                        0.94 ± 0.02              FCS/Sal × 1 6 h                                                                   --     --   --     --      0.90 ± 0.07                                                                       0.9 ± 1.2                                                                        0.84 ±             __________________________________________________________________________                                                            0.05                   Four to eight mice were used per group. Data from individual experiments      with LIFinjected mice are listed in groups with their corresponding           control mice injected with FCS/saline listed on the following line. The       frequency of injections per day is indicated: eg, × 3. Values           recorded are mean values ± SDs. The methods for deriving estimates of      spleen megakaryocytes, erythrocyte sedimentation rates (ESR), and serum       calcium/albumin ratios are detailed in Materials and Methods.                 *Values differing significantly from control values (P < .01).           

                                      TABLE 3                                     __________________________________________________________________________    LIF-Induced Changes in Marrow Spleen and Peritoneal Cell Populations of       DBA/2 Mice                                                                              Total Cells                                                                          Mean Percent                                                           (×10.sup.6)                                                                         Promyelo-                                                                           Meta-                      Nucleated                   Injected                                                                           or Spleen   cytes myelocytes                 Erythroid              Organ                                                                              With Weight (mg)                                                                          Blasts                                                                             Myelocytes                                                                          Polymorphs                                                                           Lymphocytes                                                                          Monocytes                                                                           Eosinophils                                                                          Cells                  __________________________________________________________________________    Bone LIF  11.0 ± 1.8*                                                                       4.9 ± 3.2                                                                       7.8 ± 2.6                                                                        36.3 ± 5.6*                                                                       14.0 ± 3.1*                                                                       6.8 ± 2.4                                                                        4.8 ± 2.9                                                                         25.4 ± 4.6          Marrow                                                                             FCS/Sal                                                                            18.5 ± 3.5*                                                                       2.8 ± 1.5                                                                       7.8 ± 2.7                                                                        28.5 ± 4.3*                                                                       23.8 ± 3.4*                                                                       5.6 ± 2.6                                                                        5.8 ± 2.3                                                                         25.7 ± 5.7          Spleen                                                                             LIF  107 ± 14                                                                          3.7 ± 1.4                                                                       1.9 ± 2.1                                                                         3.9 ± 2.8*                                                                        54.1 ± 11.5*                                                                     1.6 ± 1.2                                                                        0.9 ± 1.8                                                                           33.9 ± 11.2*           FCS/Sal                                                                            92 ± 14                                                                           2.7 ± 1.2                                                                       0.4 ± 0.7                                                                         1.3 ± 1.1*                                                                       77.6 ± 6.7*                                                                       1.1 ± 1.0                                                                        0.9 ± 1.0                                                                          16.0 ± 5.4*        Per- LIF  23.3 ± 15.7    3.5 ± 9.2                                                                         48.0 ± 9.5*                                                                       45.8 ± 9.9*                                                                       2.6 ± 1.5*                itoneal                                                                            FCS/Sal                                                                            13.2 ± 6.9     1.3 ± 2.2                                                                         14.4 ± 6.4*                                                                       65.7 ± 9.3*                                                                       18.3 ± 12.0*              Cells                                                                         __________________________________________________________________________     Data from 12 mice of each type examined after 14 days of injections of 2      μg LIF three times daily. Mean values ± SDs.                            *Data differing significantly (P < .01).                                 

                  TABLE 4                                                         ______________________________________                                        Effect of LIF on Megakaryocyte Progenitors                                    and Megakaryocytes                                                                   DBA/2         C3H/HeJ                                                  Parameter                                                                              LIF       FCS/Sal   LIF     FCS/Sal                                  ______________________________________                                        Megakaryocyte                                                                 Progenitors                                                                   Bone marrow                                                                            8.4 ± 3.7*                                                                           5.1 ± 2.1*                                                                           6.6 ± 2.1*                                                                         3.4 ± 2.0*                            Spleen   3.9 ± 3.7*                                                                           0.4 ± 0.5*                                                                           5.9 ± 5.1*                                                                         0.6 ± 0.5*                            Megakaryocytes                                                                Bone marrow                                                                            9.6 ± 1.3*                                                                           5.0 ± 1.1*                                                                           4.4 ± 1.0*                                                                         3.0 ± 0.7*                            Spleen   442 ± 134*                                                                           93 ± 42*                                                                             148 ± 107                                                                          76 ± 48                               ______________________________________                                         Mice were injected three times daily with 2 μg LIF for 14 days.            Progenitor cells were estimated from cultures of 2.5 × 10.sup.4         cells. Megakaryocytic numbers are number per sternal marrow segment or        spleen section corrected for area. Mean values ± SDs from eight mice o     each type.                                                                    *Indicate significantly different values (P < .01).                      

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I claim:
 1. A method for treating thrombocytopenia in a mammal whichmethod comprises administering to said mammal an effective amount ofLeukaemia Inhibitory Factor (LIF) for a time and under conditionssufficient to increase the level of formation of megakaryocytes or theirprogenitors or increase the level of platelets.
 2. The method accordingto claim 1 which further comprises administering LIF in simultaneous orsequential combination with one or more other cytokines havingmegakaryocytopoietic or thrombocytopoietic activity.
 3. The methodaccording to claim 2 wherein the other cytokine comprises at least oneof interleukin-3 (IL-3), thrombopoietin and interleukin-6 (IL-6).
 4. Themethod according to claim 3 wherein the other cytokine is IL-3.
 5. Themethod according to any one of claims 1-4 wherein the LIF and othercytokine is of human, murine or livestock origin.
 6. The methodaccording to claim 5 wherein the LIF and other cytokine are prepared byrecombinant or synthetic means.
 7. The method according to claim 1wherein the mammal is human or a livestock animal.
 8. The methodaccording to claim 1 wherein the route of administration is by theintravenous, interperitoneal, intramuscular or subcutaneous route.
 9. Apharmaceutical composition for treating thrombocytopenia, comprising aneffective amount of LIF in combination with at least one of IL-3,thrombopoietin and IL-6, admixed with a pharmaceutically acceptablecarrier.
 10. The composition according to claim 9 comprising LIF incombination with IL-3, admixed with a pharmaceutically acceptablecarrier.